Recombinant Lactobacillus plantarum UDP-N-acetylmuramoylalanine--D-glutamate ligase (murD)

What is UDP-N-acetylmuramoylalanine--D-glutamate ligase (murD) and what is its role in bacterial physiology?

UDP-N-acetylmuramoylalanine--D-glutamate ligase (murD) is a cytoplasmic enzyme essential for bacterial peptidoglycan biosynthesis. It catalyzes the addition of D-glutamate to the nucleotide precursor UDP-N-acetylmuramoyl-L-alanine (UMA) through an ATP-dependent reaction . This reaction is critical for cell wall formation and represents the second step in the assembly of the peptide moiety of peptidoglycan. The reaction proceeds according to the following mechanism:

UDP-MurNAc-L-Ala + D-Glu + ATP ⇔ UDP-MurNAc-L-Ala-D-Glu + ADP + Pi

The reaction occurs through a phosphorylation of the C-terminal carboxylate of UDP-MurNAc-L-alanine by ATP's γ-phosphate, creating an acyl phosphate intermediate. This is followed by nucleophilic attack by the D-glutamate amide group to produce the final product . The peptidoglycan layer is crucial for protecting bacteria against osmotic lysis, making murD and related enzymes attractive targets for antibacterial agent development .

What is the structural organization of murD enzyme?

The crystal structure of murD has been solved to 1.9 Å resolution, revealing a complex three-domain architecture . The enzyme comprises three domains, each resembling nucleotide-binding folds:

• N-terminal domain: Exhibits a dinucleotide-binding fold consistent with the Rossmann fold

• Central domain: Shows a mononucleotide-binding fold similar to those observed in the GTPase family

• C-terminal domain: Features another dinucleotide-binding Rossmann fold

This structural organization facilitates the binding of both the UMA substrate and ATP, which are essential for the catalytic function of murD. The structure reveals specific binding sites for UMA and allows for the identification of residues that interact with ATP through comparison with known NTP complexes . Understanding this structure is critical for designing targeted inhibitors and studying enzyme function.

How can recombinant Lactobacillus plantarum be utilized in research applications?

Recombinant L. plantarum strains have demonstrated significant potential as expression and delivery systems for vaccines targeting mucosal immunity . These bacteria can be genetically modified to express various antigens and immunomodulatory molecules. For example, researchers have developed recombinant L. plantarum strains that express:

• P14.5 protein from African swine fever virus

• P14.5-IL-33-Mus fusion proteins

• CTA1-p14.5-D-D fusion constructs

These recombinant strains not only influence gut microbiota composition but also enhance immune responses. Studies show they can increase species diversity of gut bacteria based on Shannon-Wiener index measurements and modify microbial community structures as evidenced by beta diversity analysis . The ability to express foreign proteins while naturally modulating immune responses makes recombinant L. plantarum valuable for both fundamental research and potential therapeutic applications.

What are the optimal methods for constructing recombinant Lactobacillus plantarum strains?

Construction of effective recombinant L. plantarum strains requires specific methodological approaches to ensure proper expression of target proteins. Based on recent research, the following protocol has proven successful:

• Obtain base sequences of target genes from reliable databases (e.g., NCBI) or literature

• Optimize the sequences for expression in L. plantarum

• Synthesize the optimized sequences commercially or through PCR-based methods

• Clone the target genes into appropriate expression vectors (e.g., pLP-S vector)

• Transform the constructed plasmid into L. plantarum NC8 (CCUG61730) or similar suitable strains

• Verify the recombinant strains through sequencing and expression analysis

This approach has been successfully implemented to generate recombinant L. plantarum strains expressing various fusion proteins, including NC8-pLP-S-p14.5, NC8-pLP-S-p14.5-IL-33-Mus, and NC8-pLP-S-CTA1-p14.5-D-D . When designing constructs, researchers should consider codon optimization, selection of appropriate signal peptides for secretion (if desired), and suitable promoters for optimal expression in L. plantarum.

What techniques are most effective for investigating the immunomodulatory effects of recombinant Lactobacillus plantarum?

Investigating the immunomodulatory effects of recombinant L. plantarum requires a comprehensive approach combining in vivo animal models and detailed immunological analysis. The following methodological framework has demonstrated efficacy:

• Animal model design:

  • Use genetically consistent mouse models (e.g., C57BL/6 female mice, 6-weeks old)

  • Implement controlled housing conditions with ad libitum access to food and water

  • Acclimate animals to environment for one week prior to experimentation

  • Establish appropriate control groups receiving vehicle (e.g., 0.9% normal saline)

• Immunization protocol:

  • Administer recombinant bacteria orally at defined doses (e.g., 1×10^9 CFU in 200 μL)

  • Follow prime-boost regimen: initial immunization (days 1-3), first boost (days 10-12), second boost (days 21-23)

• Immune response assessment:

  • Measure serum antibody levels (IgG, IgG1)

  • Quantify mucosal antibodies in feces (sIgA)

  • Perform flow cytometry analysis of lymphoid tissues to enumerate:

    • CD4+ T cell populations (naïve, memory, effector)

    • CD8+ T cell populations

    • B cell populations (including IgA+ B cells)

    • Monocyte and macrophage populations

• Tissue-specific analysis:

  • Conduct immunohistochemistry on target organs

  • Assess cell recruitment and inflammatory responses in tissues

This methodological approach enables detailed characterization of both systemic and mucosal immune responses to recombinant L. plantarum strains, providing insights into their immunomodulatory mechanisms.

How can researchers assess the impact of recombinant Lactobacillus plantarum on gut microbiota?

Evaluating the impact of recombinant L. plantarum on gut microbiota requires sophisticated microbiome analysis techniques. An effective methodological approach includes:

• Sample collection:

  • Collect fecal samples at defined timepoints before, during, and after administration

  • Process samples immediately or store at -80°C to preserve microbial DNA

• DNA extraction and sequencing:

  • Extract microbial DNA using specialized kits designed for fecal samples

  • Amplify the 16S rRNA gene (V3-V4 regions) using universal primers

  • Perform high-throughput sequencing (e.g., Illumina platform)

• Bioinformatic analysis:

  • Process raw sequence data through quality filtering and chimera removal

  • Classify sequences into Operational Taxonomic Units (OTUs)

  • Calculate alpha diversity metrics:

    • Shannon-Wiener index (for species diversity)

    • Observed OTUs (for species richness)

  • Perform beta diversity analysis to assess community structure changes

    • Principal Coordinate Analysis (PCoA)

    • Non-metric Multidimensional Scaling (NMDS)

• Functional prediction:

  • Use bioinformatic tools to predict metabolic and immune-regulatory functions

  • Analyze correlations between microbial changes and host immune parameters

This comprehensive approach has revealed that recombinant L. plantarum can significantly boost the species diversity of gut bacteria and modify the microbial community structure, potentially enhancing metabolic and immune-regulatory functions .

What factors should be considered when designing experiments to study murD function in recombinant Lactobacillus plantarum?

When designing experiments to investigate murD function in recombinant L. plantarum, researchers should consider several critical factors:

• Expression system selection:

  • Choose between constitutive or inducible promoters based on research objectives

  • Consider codon optimization for improved expression in L. plantarum

  • Evaluate the impact of expression levels on bacterial physiology

• Enzymatic activity assessment:

  • Develop specific assays to measure murD activity in cell extracts

  • Consider using radiolabeled substrates or HPLC-based methods

  • Include appropriate controls to account for background activity

• Structural studies:

  • Plan for protein purification with methods that maintain enzyme structure

  • Consider crystallography approaches similar to those used for E. coli murD

  • Use site-directed mutagenesis to study structure-function relationships

• Physiological impact evaluation:

  • Monitor growth characteristics under various conditions

  • Assess cell wall integrity using appropriate staining techniques

  • Measure peptidoglycan composition changes

• Experimental controls:

  • Include wild-type L. plantarum strains

  • Use recombinant strains expressing unrelated proteins

  • Implement empty vector controls

An effective experimental design should incorporate multiple approaches to provide comprehensive insights into murD function, considering both enzymatic activity and physiological impacts on the recombinant bacterial host.

How should researchers design studies to evaluate the protective effects of recombinant Lactobacillus plantarum in disease models?

Designing robust studies to evaluate protective effects of recombinant L. plantarum in disease models requires careful consideration of multiple experimental parameters:

• Disease model selection:

  • Choose models relevant to the therapeutic target

  • Consider both acute and chronic disease models when appropriate

  • Ensure reproducibility of the model

• Treatment protocol design:

  • Determine optimal dosing regimen:

    • Pre-treatment: Administer bacteria before disease induction

    • Therapeutic: Administer after disease establishment

    • Combined approach: Both pre-treatment and therapeutic administration

  • Define appropriate dose levels (e.g., 1×10^9 CFU)

  • Establish administration schedule (e.g., three consecutive days followed by boosting)

• Outcome measures:

  • Primary endpoints:

    • Disease-specific clinical parameters (e.g., weight loss, survival)

    • Pathogen burden in target tissues

    • Histopathological assessment

  • Secondary endpoints:

    • Immune response parameters

    • Microbiome composition changes

    • Inflammatory markers

• Control groups:

  • Vehicle control (e.g., saline)

  • Non-recombinant L. plantarum

  • Different recombinant constructs for comparison

This approach has proven effective in studies evaluating L. plantarum's protective effects against leptospirosis, where pre-treatment with L. plantarum significantly restored body weight in infected mice and reduced histopathological signs of disease despite not preventing pathogen access to target organs .

What analytical techniques should be employed to characterize the structural and functional properties of murD in Lactobacillus plantarum?

Comprehensive characterization of murD's structural and functional properties requires a multi-technique approach:

Previous successful structural characterization of murD employed multiple anomalous dispersion using the K-shell edge of selenium combined with multiple isomorphous replacement to solve the crystal structure at 1.9 Å resolution . This multi-technique approach provides comprehensive insights into both structure and function, facilitating deeper understanding of murD's role in peptidoglycan biosynthesis.

How should researchers interpret immunological data from studies using recombinant Lactobacillus plantarum?

Interpreting immunological data from recombinant L. plantarum studies requires systematic analysis considering multiple factors:

• Antibody response interpretation:

  • Compare systemic (serum IgG, IgG1) and mucosal (fecal sIgA) antibody levels between treatment groups

  • Consider both magnitude and kinetics of antibody responses

  • Correlate antibody responses with protective efficacy in challenge models

• Cellular immune response analysis:

  • Examine changes in lymphoid tissue immune cell populations:

    • Increased B cells indicate enhanced humoral immunity

    • Shifts in CD4+ T cell subsets (naïve to effector) suggest active immune response

    • Changes in CD8+ T cell populations reflect cell-mediated immunity development

    • Monocyte/macrophage recruitment indicates innate immune activation

• Tissue-specific immune response evaluation:

  • Analyze immunohistochemistry data to determine:

    • Neutrophil and macrophage recruitment to affected tissues

    • Reduction in total leukocytes and T cells in tissues like kidney

    • Correlation between immune cell profiles and histopathological findings

• Contextual interpretation:

  • Consider baseline immune status before intervention

  • Account for strain-specific effects of L. plantarum

  • Evaluate recombinant protein expression levels and stability

Recent studies demonstrate that recombinant L. plantarum treatment leads to increased IgG and IgG1 in serum and sIgA in feces, along with enrichment of CD4+ T cells and IgA+ B cells, indicating complex immunomodulatory effects . Similarly, pre-treatment with L. plantarum in a leptospirosis model showed significant immune cell profile changes, including increased B cells and shifts in T cell populations from naïve to effector phenotypes .

What statistical approaches are most appropriate for analyzing microbiome data in recombinant Lactobacillus plantarum studies?

Microbiome data analysis in recombinant L. plantarum studies requires specialized statistical approaches to account for the complex, high-dimensional nature of microbiome datasets:

• Alpha diversity analysis:

  • Apply Shannon-Wiener index for species diversity comparisons

  • Use appropriate statistical tests (e.g., Wilcoxon rank-sum test, ANOVA with post-hoc tests)

  • Consider correction for multiple comparisons (e.g., Bonferroni, FDR)

• Beta diversity analysis:

  • Implement distance metrics (e.g., Bray-Curtis, UniFrac)

  • Apply ordination techniques (PCoA, NMDS)

  • Test significance using PERMANOVA or ANOSIM

  • Create clear visualization of community structure shifts

• Differential abundance analysis:

  • Use specialized methods designed for microbiome count data:

    • DESeq2

    • ANCOM

    • ALDEx2

  • Apply appropriate transformations for non-normal distributions

  • Control for false discovery rate in multiple comparisons

• Correlation and network analysis:

  • Correlate microbial changes with host parameters

  • Generate co-occurrence networks to identify microbial interactions

  • Apply appropriate correlation metrics (Spearman's rank for non-normal data)

Research has shown that recombinant L. plantarum can dramatically boost gut bacterial species diversity and alter microbial community structure, with these changes correlating with enhanced functions in metabolism and immune regulation . Proper statistical analysis is crucial for accurately interpreting these complex relationships.

How can researchers effectively correlate enzymatic activity of murD with physiological outcomes in recombinant Lactobacillus plantarum studies?

Establishing meaningful correlations between murD enzymatic activity and physiological outcomes requires methodical approaches to data integration:

• Quantitative enzyme activity measurement:

  • Develop standardized assays to quantify murD activity in cell extracts

  • Measure activity under various physiological conditions

  • Consider using purified enzyme for baseline comparisons

• Physiological parameter assessment:

  • Monitor growth characteristics (lag phase, doubling time, maximum density)

  • Evaluate cell morphology and integrity

  • Measure peptidoglycan composition and structure

  • Assess stress resistance (osmotic, acid, bile salt)

• Correlation analysis:

  • Apply regression analysis to identify relationships between enzyme activity and physiological outcomes

  • Consider non-linear relationships and threshold effects

  • Account for confounding variables in experimental design

• Validation approaches:

  • Use site-directed mutagenesis to create variants with altered activity

  • Implement inducible expression systems to modulate enzyme levels

  • Apply enzyme inhibitors at varying concentrations

• Data integration:

  • Create integrated models incorporating enzymatic, structural, and physiological data

  • Use multivariate analysis to identify patterns and relationships

  • Develop predictive models of physiological outcomes based on enzyme activity

This systematic approach enables researchers to establish causal relationships between murD activity and bacterial physiology, providing insights into peptidoglycan synthesis regulation and potential targets for intervention.

What are the current limitations in studying recombinant Lactobacillus plantarum expressing murD?

Several significant challenges currently limit research on recombinant L. plantarum expressing murD:

• Expression system constraints:

  • Limited availability of well-characterized expression vectors for L. plantarum

  • Challenges in achieving high-level expression without affecting bacterial fitness

  • Difficulty in controlling protein localization (cytoplasmic vs. secreted)

• Structural and functional analysis limitations:

  • Challenges in purifying sufficient quantities of properly folded enzyme

  • Limited availability of species-specific antibodies for detection and quantification

  • Technical difficulties in crystallizing membrane-associated enzymes

• In vivo research challenges:

  • Variability in colonization efficiency of recombinant strains

  • Difficulty distinguishing direct effects of murD from secondary immunomodulatory effects

  • Challenges in maintaining stable expression in the absence of selection pressure

• Translational barriers:

  • Regulatory considerations for genetically modified organisms in clinical applications

  • Scaling issues for consistent production of recombinant strains

  • Stability concerns during formulation and storage

Addressing these limitations requires interdisciplinary approaches combining molecular biology, structural biochemistry, immunology, and bioinformatics to develop improved expression systems, analytical methods, and experimental models.

How might recombinant Lactobacillus plantarum murD research contribute to novel antimicrobial strategies?

Recombinant L. plantarum murD research offers several promising avenues for developing novel antimicrobial strategies:

• Target-based drug discovery:

  • Detailed structural studies of murD can identify unique features for selective inhibitor design

  • Comparison of murD across bacterial species can reveal conserved and variable regions

  • Structure-based virtual screening can identify potential inhibitors for experimental validation

• Probiotic enhancement strategies:

  • Engineering L. plantarum to modulate murD expression may enhance cell wall integrity

  • Improved stress resistance could enhance probiotic survival and colonization

  • Optimized peptidoglycan composition might enhance immunomodulatory properties

• Vaccine delivery platforms:

  • Recombinant L. plantarum expressing murD-antigen fusions could serve as mucosal vaccines

  • Co-expression with immunomodulatory molecules (like IL-33) could enhance vaccine efficacy

  • Targeting of specific immune cell populations could be achieved through engineered constructs

• Competitive exclusion strategies:

  • Engineered L. plantarum with optimized murD function could enhance gut colonization

  • Displacement of pathogenic bacteria through competitive exclusion

  • Modulation of gut microbiota toward beneficial compositions

The peptidoglycan biosynthetic pathway represents an attractive target for antibacterial development , and recombinant L. plantarum research provides unique opportunities to exploit this pathway while leveraging the probiotic and immunomodulatory properties of this beneficial microorganism.

What innovative research directions might emerge from combining murD studies with immunomodulatory applications of Lactobacillus plantarum?

The integration of murD research with L. plantarum immunomodulatory studies presents several innovative research directions:

• Engineered immunotherapeutics:

  • Development of recombinant L. plantarum strains with modified murD to optimize peptidoglycan presentation to immune cells

  • Creation of strains co-expressing murD with immunomodulatory molecules for enhanced effect

  • Design of murD-antigen fusion proteins for targeted immune responses

• Microbiome-immune interaction studies:

  • Investigation of how murD-mediated changes in peptidoglycan affect microbiota composition

  • Examination of relationships between altered peptidoglycan structure and immune cell activation

  • Exploration of how these changes influence gut-brain axis signaling

• Precision probiotic development:

  • Engineering of L. plantarum strains with optimized murD function for specific disease conditions

  • Tailoring of peptidoglycan composition to induce particular immune responses

  • Development of condition-specific probiotics based on murD modification

• Multi-functional therapeutic platforms:

  • Creation of recombinant strains combining optimized murD function with:

    • Production of therapeutic proteins or peptides

    • Expression of disease-specific antigens

    • Delivery of RNA-based therapeutics

• Novel adjuvant development:

  • Exploration of modified peptidoglycan structures as mucosal adjuvants

  • Investigation of synergistic effects between peptidoglycan components and other immunomodulators

  • Development of adjuvant systems with reduced inflammatory potential

Recent studies demonstrating that recombinant L. plantarum can enhance gut bacterial diversity, alter microbial community structure, and modulate immune responses provide a foundation for these innovative research directions .